The present invention relates to methods and products for controlling the expression of genes immobilized on a temperature controlled template. More particularly, the invention relates to the use of an in vitro method and apparatus for controlling gene expression.
Gene expression is controlled by a variety of regulatory mechanisms (Alberts B, et al. (1994) xe2x80x9cMolecular Biology of the Cellxe2x80x9d New York, Garland). Advances in genome sequencing (Olson M. V. (1993) Proc. Natl. Acad. Sci. 90:4338-4344) and DNA chips (xe2x80x9cDNA chipsxe2x80x9d (1999) Special Issue, Nature Genetics, suppl. vol. 21, January, 1999) provide insight into the collective gene expression pattern and protein function that control networks in cells.
Protein production is a result of a two step process: transcription and translation. First RNA polymerase (RNAP) transcribes DNA into messenger RNA (mRNA) and subsequently a ribosome translates mRNA into a protein. During translation, the protein is in a stable complex with mRNA via the ribosome. At the end of translation, when the ribosome reaches a stop codon, the newly synthesized protein is released from the ribosome as well as the mRNA. Similarly, in transcription, the mRNA stays associated with the DNA via RNAP, and is released when the polymerase reaches a terminal signal.
For translation, the stop codon used are UAA, UGA or UAG. Experimentally one can delete the stop codon function. In one method a piece of DNA is annealed complementary to the sequence along the coding sequence (between start and stop codon). Under such conditions, the translation stalls at the annealing site, and the protein remains in complex with the ribosome and mRNA (Haeuptle, et al. (1986) Nucleic Acids Res 14(3):1427-48). In another approach, the stop codon is deleted from the initial DNA. Without stop codon, the ribosome will move along the mRNA to the very last base, but will not release the protein. Such xe2x80x9cstoplessxe2x80x9d coding has been used in experiments where a stable attachment of protein to the mRNA is required, for example, in vitro protein evolution (He et al. (1997) Nucleic Acids Res. 25(24):5132-5134) and co-translational protein folding (Fedorov et al. (1995) Proc. Natl. Acad. Sci. USA 92(4):1227-1231; Makeyev, et al. (1996) FEBS Lett 378(2):166-170).
Normally the signal to terminate transcription is a terminator sequence on the DNA. The signal, in vitro, can also be the very end of a linear DNA. This end-of-DNA termination signal is commonly used in run-off transcription, where RNAP runs to the end of DNA and falls off from it. There are methods to prevent RNAP from reaching the termination signal, for example, by covalently crosslinking the DNA duplex structure (Shi, et al. (1988) J. Biol. Chem. 263(1):527-534; Shi, et al. (1988) J. Mol. Biol. 199(2):277-293), or by DNA lesion (Sauerbier et al. (1978) Annu. Rev. Genet. 12:329-363; Mellon et al. (1989) Nature 342(6245):95-98; (Selby et al. (1993) Science 263(1):527-534). Under these conditions, mRNA remains attached to the DNA via RNAP.
The human genome is the genetic material in human egg and sperm cells which contain 3xc3x97109 base pairs (bp) of DNA. The sequence of 3xc3x97109 bp corresponds to 750 megabytes of information. If the sequence of the human genome could be determined, it would be possible to store and manipulate it on a personal computer. Olson (1993) Proc. Natl. Acad. Sci. USA 90:4338-4344. Refinements in experimental protocols, instrumentation and project management have made it practical to acquire sequence data on an enlarged scale. Id. Once genomic sequences are known any gene construct is easy to implement. Once function is understood it becomes possible to realize in vitro protein networks (Jermutus et al. (1998) Curr. Op. Biotechnology 9:534-548) similar to the biological ones.
Arrays offer a systematic way to survey DNA and RNA variation. Lander (1999) Perspective:Nature Genetics, suppl: 1-8. Arrays offer opportunities to analyze large numbers of sequence interactions. Southern et al. (1999) Nature Genetics (supp) 21:5-9. Array-based methods of observing DNA hybridization to complementary RNA are known in the art. Id.
The present invention is directed to the use of micron scale heaters for gene expression. The present invention is based on the unique determination that in vitro temperature controlled protein expression is achievable on a micron scale.
The micron scale heaters and methods of the present invention are useful for preparing in vitro programmable protein networks and protein micro arrays. In accordance with the present invention protein micro arrays are synonymous with biological chips or DNA chips. The products and methods of the present invention are particularly useful for comparative expression analysis, the analysis of molecular interactions and for providing insights into complex biochemical networks.
One embodiment of the present invention provides an apparatus for controlling gene expression comprising a temperature-controlled template having a nucleic acid construct immobilized thereon. The temperature-controlled template comprises a metal oxide pad with attached electrodes. In a preferred embodiment the metal oxide pad is an indium tin oxide (ITO) pad.
The metal oxide pad is affixed to a substrate, such as glass, for example. The metal oxide pads contain avidin-coated beads, which bind to biotinylated nucleic acid constructs. The metal oxide-substrate combination of the present invention is mounted on a water-cooled brass sample plate. The apparatus also optionally includes a means for detecting protein expression.
Another embodiment of the present invention provides an in vitro programmable protein micro array. Such array includes a plurality of temperature-controlled metal oxide pads (e.g. ITO pads) mounted on a substrate (e.g. glass) wherein each pad is individually temperature-controlled. Each temperature-controlled metal oxide pad comprises at least one immobilized nucleic acid construct of a specific type which is capable expressing a protein which is localized with the nucleic acid construct.
In another embodiment, the present invention provides an in vitro programmable protein network having a plurality of temperature-controlled metal oxide pads (e.g. ITO) mounted on a substrate (e.g. glass) wherein each pad is individually temperature-controlled. Each temperature-controlled metal oxide pad comprises at least one immobilized nucleic acid construct of a different type which is capable expressing a protein which is released from the nucleic acid construct.
The present invention also provides an in vitro method of controlling gene expression by immobilizing a nucleic acid construct on a temperature-controlled template, applying a cell extract, expressing a protein and detecting the expression of the protein.